J. Cell Sci. 12, 287-311 (1973) 287 Printed in Great Britain THE SPERMATOZOON OF ARTHROPODA XVIII. THE NON-MOTILE BIFURCATED SPERM OF PSYCHODIDAE FLIES B. BACCETTI, R. DALLAI AND A. G. BURRINI Institute of Zoology of the University of Siena, Via Mattioli 4, Siena, Italy SUMMARY The spermatozoa of some species of Diptera belonging to the Psychodidae are described. In the 4 species examined, they appear needle-shaped, non-motile, anteriorly flattened, posteriorly bifid. Flagella, and any microtubule system, are lacking, and the only organelles encountered are an extremely elongated nucleus, a prominent acrosome, a well developed subacrosomal body, a slender, somewhat elongated mitochondrial derivative and a conspicuous multilayered membrane wall, produced by the superimposition of the plasma membrane and the acrosomal membrane. The mitochondrion is devoid of a crystalline axis, while the acrosome contains a longitudinal, cross-striated para-crystalline body. Centrioles are absent throughout the spermatid stage. Sperms and spermatids are organized in syncytia of 210 elements. INTRODUCTION So far, a non-motile sperm type among insects has not been reported. In the few instances in which the classic axoneme is absent, it is replaced either by a larger number of doublet bundles, as in Protura (Baccetti, Dallai & Fratello, 1973) or the dipteran Sciara (Phillips, 1966; Makielski, 1966), or else by a bundle of simple micro- tubules repeatedly coiled around the sperm, as in coccid insects (Robison, 1966, 1970; Ross & Robison, 1969; Ross, 1971). The sperm morphology of high Isoptera is still unknown, and will be tackled by us in the near future. The present investigation deals with the hitherto undescribed spermatozoon of Diptera belonging to the Psycho- didae which appears to lack an axoneme, to be non-motile and caudally bifid. MATERIALS AND METHODS Specimens of Telmatoscopus albipunctatus, Psychoda cinerea, Psychoda sp. and Psychoda alternata were supplied in all the instars by Drs M. Sara and G. Salamanna of the Institute of Zoology of the University of Genova. Larvae, pupae and adults were dissected out in cold 5 % glutaraldehyde at pH 7-2 to 7-4 in Hoyle buffer containing 5% sucrose; the testes were excised and fixed in the same fluid for 1-5 h, rinsed overnight in Hoyle buffer with 5 % sucrose, postfixed in 1 % osmium tetroxide for 45 min, dehydrated in a graded series of ethanols and embedded in Epon. Sections, cut with an LKB ultramicrotome, mounted on grids, and stained with uranyl acetate and lead citrate, were observed and photographed under the Siemens Elmiskop IA and Philips EM 300 electron microscopes. For study of proteins, thin sections were oxidized for 5 min in H2O2, 12 vol., thoroughly rinsed in distilled water and treated with 0-2% pepsin solution in o-i N HC1 for 8-15 min at 37 °C. Control specimens were incubated in o-i N HC1 for the same 288 B. Baccetti, R. Dallai and A. G. Burrini periods as the experimentals. The subcellular localization of polysaccharides was investigated by the periodic acid-thiosemicarbazide-silver proteinate method (Thiery, 1967) with 24-36 h action of TCS. To evaluate sperm motility, sperm cells, suspended between 2 slides in Gouldin medium, were observed under a Leitz Ortholux phase-contrast microscope. Stereoscopic examination was carried out by the JSM-2 scanning electron microscope on spermatozoa obtained by squeezing the testes, coated with evaporated gold/platinum. For freeze-etching the testes were incubated for 1 h at 40 C in 25 % glycerol in Hoyle buffer, transferred on to small copper disks and rapidly plunged in Freon 23 cooled with liquid nitrogen for 1 min. The disks were then immersed in liquid nitrogen for a period of 10 min to 3 days. Freeze-etching was achieved with a Balzers apparatus, according to the technique devised by Moor, Miihlethaler, Waldner & Frey-Wyssling (1961). The carbon-platinum replica was removed from the specimen holder, treated with chromic acid, thoroughly rinsed in distilled water in order to remove cellular and other debris, and then picked up on copper grids. RESULTS The sperm examined under fresh conditions (Fig. 3) by phase-contrast microscopy are needle-shaped, about 0-2 mm long. When maintained in fluids which as a rule allow considerable motility in other insect spermatozoa, among Diptera as well, they appear altogether non-motile, nor can they be stimulated in any way by changing the environmental conditions. After coating with gold-platinum, examination in the scanning electron microscope reveals morphological characteristics of particular interest. The anterior portion of the sperm (Fig. 4), about 22-5 /un long, looks markedly flat in all the species, except Telmatoscopus, where it appears keeled lengthwise. In every case, the posterior portion is consistently bifid (Fig. 5) along the terminal 3 /tm of its length. For most of the remaining intermediate portion the sperm is cylindrical. Ultrathin sections make it possible both to confirm these peculiarities and clarify the structure of the sperm. In the 4 species examined here, the sperm is non-flagellated and devoid of any microtubule system whatsoever. The organelles encountered in the mature sperm are the nucleus, a prominent acrosome, a strikingly developed sub- acrosomal body, a slender, somewhat elongated mitochondrial derivative and a con- spicuous multilayered membrane wall surrounding the whole sperm cell and also separating the nucleus from the other organelles. In Telmatoscopus albipunctatus the nucleus lies in a dorsal position beneath the keel of the anterior part of the sperm and along its major cylindrical portion. It is moder- ately compact, its chromatin being distributed into filaments which are arranged longitudinally, and always readily identifiable. The nucleus appears as a cylinder (Fig. 10) which gradually flattens out rostralwards, appearing first elliptical (Fig. 9), then C-shaped in cross-section, with dorsally directed convexity (Fig. 8), and finally, at the anterior end of the sperm, presents a thin crescent shape in profile. At this level, the nucleus is seen to expand across almost the whole width. In the posterior regions of the sperm, the nucleus appears as a cylinder with gradually reduced diameter, still lying in a dorsal position (Fig. 11). Ventrally, the sperm is almost entirely occupied by the acrosome, which extends Non-motile bifurcated sperm of Psychodidae 289 distally from the rostral end (Fig. 7), where, reniform in section, it is the only organelle present, to the bifid tail (Fig. 13), filling up the empty spaces left by the other organ- elles (Fig. 11). The acrosome consists of a compact, granular material particularly packed along its axis which is positioned in the centre of the cell. This axis (Fig. 12) exhibits a clear transverse banding, with a period averaging 25 nm. Each period con- sists of a highly electron-dense band and 2 moderately electron-dense bands, which are separated by an interval some 7 nm wide and lie 10 nm from the major band. This striated material is undoubtedly protein in nature, despite its pronounced pepsin- resistance. In the other region, the acrosome material is not striated, and is pepsin- sensitive, as shown in the posterior segment of the spermatozoon, where it represents almost the whole content (Fig. 15). The nucleus is separated from the acrosome by a conspicuous subacrosomal structure (Fig. 10) simply represented by a furrow about 40—50 nm wide, which extends almost the whole length of the nucleus, assuming the ventral shape of the latter. Along this furrow the nuclear envelope and the acrosomal membrane are likely to fuse. This structure undergoes a marked thickening just in front of the flat anterior region of the sperm (Fig. 9), taking on an ellipsoidal outline first, then an almost circular one in cross-section. At both these levels the subacrosomal structure fills up the sperm almost completely, since dorsally the nucleus is thin and C-shaped, as is the acrosome ventrally. The latter also extends within the 2 wings of the spermato- zoon. In the posterior segment of the spermatozoon the subacrosomal material sur- rounds the nucleus, and proceeds beyond it in caudal direction in a short region, where it appears as a clear sector in the opaque circle constituting the acrosome (Fig. 14). The subacrosomal material is granular, homogeneous, and fairly transparent to the electron beam. It is readily removed by pepsin digestion (Fig. 16), being exclusively protein in nature. It is Thiery-unreactive (Fig. 17) and a-amylase-resistant. Within the acrosome, the mitochondrial derivative is found. It is narrow, elongate, 130 nm wide and some 60 nm thick in sectional view, with its concavity facing the nucleus. In both transverse and longitudinal sections it appears devoid of cristae; its matrix is highly transparent to the electron beam. The mitochondrial derivative is the smallest sperm component. In fact, arising behind the rostral flattened region where the sperm is elliptical in cross-section, it extends only a short distance, i.e. the length of the sperm central portion. The spermatozoon is entirely bounded by a 7-layered wall (Fig. 10) whose overall thickness is 25 nm. The outermost layer, 13 nm thick on average, is made up of 2 osmiophilic laminae divided by a light interspace; farther inwards, after a gap about 5 nm wide, a thinner membrane, averaging 7-5 nm, is encountered. In frozen-etched preparations, the aspect of these membranes stands out sharply (Figs. 18-20). Both the outer and inner aspects of the outermost membrane (13 nm thick) appear smooth, although some irregularity results from the presence of minute, randomly scattered indentations. On the other hand, the thinner membrane (7-5 nm thick) is obliquely striated, with a period of about 30 nm. The striations probably occupy the cleft 5 nm wide between the thick outermost membrane and the inner thinner one; the latter exhibits an even surface on either side, only lightly indented by scanty and widely 19 CE L 12 290 B.